Micromanipulator for a cryomicrotome

ABSTRACT

A microtome system has a cryomicrotome with a sectioning device in which is provided a preparation holder and a knife edge that are guided past one another inside a working space during a sectioning operation, in order to produce thin sections of a preparation retained in the preparation holder. A micromanipulator is operable outside the working space. With the micromanipulator, a tool for retention of a specimen support is positioned proximate the knife edge during a sectioning operation in order to receive the sections that are produced, preferably for substantially stationary retention of the specimen support.

The invention relates to a microtome, in particular a cryomicrotome,having a sectioning device in which are provided a preparation holderand a knife edge that are guided past one another inside a working spaceduring a sectioning operation, in order to produce thin sections of apreparation retained in the preparation holder.

BACKGROUND

The production of thin sections of cryopreserved specimens, inparticular biological specimens with vitrified material (i.e. solidifiedin glassy fashion), is important for cryoelectron microscopy and similarinvestigation methods. Cryopreserved specimens largely avoid distortionsthat can derive from desiccation, chemical modifications (includingcontrast agents), and other specimen stabilization methods and thereforeenable investigation of the specimen at the ultrastructure scale in astate that comes very close to the living state of the initial specimen.A very thin section thickness is required for investigation using anelectron microscope or the like, however, namely tens to a few hundrednanometers depending on the type of specimen. This, along with thedeep-frozen state (generally below −100° C.) of the sections, placesserious demands on the user's skill and on the quality and precision ofthe sectioning device.

In addition to the actual sectioning operation, removing the sectionsfrom the edge of the knife and applying them onto a specimen carrier(referred to here as a specimen support) constitutes a particularlydelicate operation. The specimen supports used for an electronmicroscope are generally grids made of a thin metal foil. Forroom-temperature applications the possibility exists, for example, ofcarrying the sections away from the edge on the surface of a water bathin which the sectioning apparatus is partly immersed; this offers theadditional advantage that sections crimped during the sectioningoperation can relax again (decompress) on the water surface, and areonly then received by the grid. The water-bath approach is not availablefor low-temperature applications, and transport from the knife edge tothe grid must therefore be effected in some other manner.

Because manipulation of sections in a cooled preparation chamber of anultramicrotome is performed manually, especially during the sectioningoperation, such manipulation requires an experienced user. Because thesections are produced in a sequence of many sectioning motions ratherthan individually, the sections are produced in the form of aninterconnected strip of a plurality of individual sections. Aninterconnected strip of this kind is then pulled with a suitable tool,for example a hair mounted at the end of a wooden rod, from the knifeedge over a grid and then immediately laid down. Accurate positioning ofthe grid with respect to the knife blade is necessary so that thefragile section strip can be picked up close to the knife edge.

A known device for producing low-temperature thin sections is the LeicaEM FC6, available from the assignee of the present application, LeicaMicrosystems, GmbH, of Hernalser Hauptstrasse 219, A-1170 Vienna,Austria. The sectioning operation takes place in a working space with atleast one side wall that is adjacent or thermally coupled to a coolingchamber fillable with a coolant (e.g. liquid nitrogen), or where theworking space is surrounded on several sides (e.g. in cupped fashion) bythe cooling chamber. The working space cooled in this fashion is opentoward the top and thus accessible to the user. The specimen to besectioned is mounted on a specimen holder positioned on one verticalside of the working space, to which holder a vertical motion can beimparted in the manner of a vibrating head. The sectioning knife isunmoved during the sectioning operation and is positioned by means of aknife holder mounted in the floor of the working space (shifting foralignment purposes is possible). The knife holder furthermore containsinstruments for retaining a grid (or a few grids), in particular a gridholder into which the grid can be clamped at its edge. This grid holderis shiftable in a horizontal direction toward and away from the knife;positioning consists solely in sliding toward the knife. An end stopprevents contact between the grid and the knife. The specimenpreparation device located in the working space can furthermore comprisean apparatus having a preparation surface, in which grids are set inplace and pressed while still in the working space. Placement of thegrid onto a preparation surface of this kind, in particular theoperation of pressing the sections onto the grid (specimen support),can, however, generate contaminants.

In order to counteract electrostatic charging of the sections in thecooled working space, the Leica EM FC6 provides an ionizer that slightlyionizes the gases (vapors) surrounding the material to be sectioned, inorder to ensure dissipation of electrical charges.

With other, earlier models, holders for multiple specimen supports wereprovided. These holders had a coarse vertical displacement capability,and (by shifting) a coarse displacement capability in the horizontalplane. These models, as well, comprised a section press integrated intothe specimen preparation device.

The aforesaid known devices have the following disadvantages:

-   -   Guidance of the section strip and positioning of the grid are        performed manually, and are thus susceptible to operating        errors, and require considerable user skill.    -   The numerous manipulation steps on the sections within the        working space are laborious and can result in section losses and        contamination.    -   The grid is retained on the side facing the knife edge. This        impedes placement of the section strip, since the section strip        is pulled from the knife edge toward the holder.    -   The effect of the ionizer is impaired by components made of        solid metal for retaining the grid.    -   There is a risk of contaminating the sections with condensed ice        or other contaminants from the environment as a result of        manipulation that lasts too long.

The article “Vitreous cryo-sectioning of cells facilitated by amicromanipulator,” by M. Ladinsky et al., J. Microsc. 224 (2006) 129-134describes a cryomicrotome arrangement having a micromanipulator. In it,a fiber retained by the micromanipulator at the end of a wooden rodserves to pick up the sections and guide the section strip to thesupport. The manipulator is mounted on the equipment table, not on thecooling chamber itself. Because a vibration damping system is presentbetween the equipment table and cooling chamber, relative motions occurbetween the manipulator and the ultramicrotome with cooling chamber, inparticular, contact by the user (e.g. operation of the microtomearrangement's stereomicroscope) inevitably causes such relative motions.

DE 202 21 696 U1 describes a microscope arrangement having amicromanipulator for carrying out microscopic manipulations andinjections on living material. The micromanipulator is attached to themicroscope by means of an adapter. DE 1279 368 A describes amicromanipulator for moving and producing small tools under amicroscope, the micromanipulator being arranged, by means of a magnetictool holder, at the lower objective end of the microscope.

SUMMARY OF THE INVENTION

Accordingly, one object of one or more embodiments of the invention isto improve the manipulation of sections immediately after the sectioningoperation. In particular, sources of error in the context of manualhandling are to be precluded.

This and other objects may be achieved by a microtome, in particular acryomicrotome, having a micromanipulator with which, according to thepresent invention, a tool for retaining a specimen support that isprovided for reception of the sections that are produced is positionableproximate the knife edge during a sectioning operation, themicromanipulator being operable outside the working space.

The micromanipulator according to the present invention enablessubstantially improved, precise positioning of the specimen holderduring the operation of loading the specimen holder with the sections. Afurther advantage of an embodiment of the present invention describedherein is that a grid loaded with sections can be laid into a transfercontainer without having to perform a removal of the retaining tool fromthe micromanipulator, by way of a positioning motion of the manipulator.

With regard to the numerous manipulation steps involving the sectionsinside the working space (position specimen and knife before cutting,pick up and position grid with holder, place section strip on grid, opengrid holder, transfer to grid press, manually pick up grid with forcepsand load into a transfer container), an embodiment of the inventiondescribed herein thus results in a definite simplification and in moreexpeditious execution of the procedure when manufacturing preparationsections, thus also reducing the risk of section losses andcontamination.

The procedure that can be carried out with the device according to suchembodiment of the present invention is not only simpler but alsosubstantially faster than with conventional units. The tool, e.g. aforceps, can be loaded with a prepared grid and, shortly beforesectioning begins, installed into the micromanipulator and positionedinto the working chamber.

A further advantage of such embodiment is that influence on the ionizeris reduced. The tool retained by the manipulator projects only with itstip into the working space. In particular, a forceps having thin tips,or even a ceramic coating or ceramic tips, can minimize the negativeinfluence on ionizer effectiveness. This yields a considerableimprovement over known devices, in which complex apparatuses havinglarge metallic surfaces are housed in the working space.

In accordance with the invention, the micromanipulator is used toposition the specimen support (instead of picking up the sections or thesection strip and delivering them/it to the specimen support). Once thespecimen support has been loaded with sections, the arrangementaccording to an embodiment of the present invention particularlydescribed herein allows the specimen support to be placed in a transfercontainer without removing the forceps from the holder by way of thepositioning motion of the manipulator. In this regard, themicromanipulator is preferably set up for substantially stationaryretention of the specimen support.

A preferred embodiment of the invention refers to a cryomicrotome havinga cooling chamber that surrounds the working space but permits the useraccess to the working space, for example through an opening (or several)that preferably allows access from above, the micromanipulator engagingthrough the opening (or one of the openings) and being operable fromoutside the cooling chamber. It is favorable in this context, in orderto exclude shocks that could lead to a relative motion between the toolretained by the micromanipulator and the material being sectioned in theworking area, if the micromanipulator is mounted on the cooling chamber.

For similar reasons, it can be advantageous to connect themicromanipulator fixedly to the microtome (e.g. on its housing or onanother load-bearing component).

In order to improve adjustment capabilities for different specimensizes, and for simplified transport of the specimen support to thetransfer container, one preferred embodiment of the micromanipulatorcomprises a three-axis positioning device.

In a preferred refinement of the invention, the micromanipulatorcomprises a receptacle for a forceps. A forceps whose tip is bent can beprovided, in order to retain the specimen support almost parallel to theknife edge. The angle of this bend is, usefully, the angle at which thelongitudinal axis is tilted with respect to the feed direction of theknife edge.

Provision can further be made that an elongated tool retained in themicromanipulator is rotatable about its longitudinal axis. This resultsin more-flexible handling of the specimen support, which can beadvantageous for placement of the grid. In addition, it permits easierhandling of the specimen holder upon transport into a transfercontainer.

In addition, a transfer container that comprises at least one receivingopening for specimen supports can be replaceably insertable in theworking space. In this case, it is advantageous if the range of motionof the micromanipulator is sufficient to move a specimen supportretained thereby to the location of the receiving opening(s) of aninserted transfer container.

Lastly, the fact that one or more axes of motion of the micromanipulatorare motorized can represent an additional improvement in operatingconvenience, in an embodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention, together with further advantages, is explained in greaterdetail below with reference to a non-limiting exemplifying embodimentthat is depicted in the appended drawings, in which:

FIG. 1 is a perspective view of an ultramicrotome having a manipulatoraccording to an embodiment of the invention;

FIG. 2 is a detail of FIG. 1 showing the manipulator and the workingspace of the ultramicrotome; and

FIGS. 3 and 4 are enlarged details of FIG. 2 of the working space,respectively showing the retained tool with a specimen support in twodifferent positions, namely during the pickup of sections (FIG. 3) andupon introduction of the specimen support into a transfer container(FIG. 4).

Repeat use of reference characters in the present specification anddrawings is intended to represent same or analogous features or elementsof one or more embodiments of the invention.

DETAILED DESCRIPTION

Reference will be made in detail to one or more embodiments of thepresent invention, one or more examples of which are illustrated in theaccompanying drawings.

FIG. 1 shows an ultramicrotome 1 that is based on the above-describedLeica EM FC6 and, in a manner known with regard to that device,comprises a cooling chamber 2 having a working chamber 3 surrounded bythe cooling chamber, in which the tools for the production of sectionsare housed. A stereomicroscope 4 serves, in a manner also known, forobserving the preparation and the sections during sectioning andsubsequent manipulation. In a departure from known devices, amanipulator 5 is installed on one side of the cooling chamber. As arule, manipulator 5 is located on the left side in order to enablemanual manipulation of the specimens from the right side of the device,although installation on the right side is, of course, also possible(especially for left-handers).

Manipulator 5 is shown in further detail in FIG. 2. Manipulator 5,constituting a micromanipulator, possesses displacement capabilities 51,52, 53 in three axes, namely 51 and 52 for horizontal motion and 53 forvertical displacement. Each axis is made up of a linear guide and amicrometer screw as displacement element. Displacement elements 54 canalso be embodied in motorized fashion by means of a precision steppingmotor. All these displacement motions act on a manipulator body 55.Located therein is a receptacle 56 for a tool 57 that can be, forexample, a forceps. Receptacle 56 is additionally rotatable about thelongitudinal axis of tool 57. With the aid of the manipulator, a grid 6retained in tool 57 can be positioned proximate a knife 7 and of thepreparation (not shown) retained in sectioning head 8. An ionizer 9,which is preferably directed toward the edge of knife 7, canadditionally be provided.

FIG. 3 shows, in a detail of FIG. 2, the manipulation of forceps 10 andof a grid 6 serving as a specimen support. The forceps can preferably beequipped with a slide 11 for immobilizing the forceps tips when clampinga grid 6. Forceps 10 is bent near the tip at an angle of approximately45°, enabling grid 6 to be positioned parallel to knife edge 71. Thisarrangement is very well suited for placement of the section strip ontogrid 6.

The arrangement of manipulator 5 and forceps 10 on one side (in thiscase the left side) of the device advantageously allows the user toaccess the working space from the other side (in this case from theright). The user can thus intervene manually during the sectioningoperation, in particular in order to remove from knife blade 71—forexample, by means of a fiber (e.g. hair) retained at the end of a stickor another holder—the section strip that is produced, and guide it to adesired destination, in this case grid 6 prepared as a specimen carrieror support. During this operation, grid 6 remains substantiallystationary, since the positioning of grid is set by means of themanipulator before the sectioning operation and then remains the sameexcept for any slight position corrections (realignments).

After deposition of the section strip (not shown) onto grid 6, thelatter needs to be brought into a transfer container. This operation canlikewise be carried out with manipulator 5 according to thepresently-described embodiment of the present invention without removingthe forceps from its holder.

FIG. 4 illustrates introduction into the transfer container, in adepiction corresponding to FIG. 3.

A transfer container 12 can be inserted, as shown, at the bottom ofworking space 3, for example alongside knife 7 or alongside the verticalplane constituted by the cutting head and knife. The transfer containercomprises a number of openings 13, e.g. four openings, which have, e.g.,a diamond-shaped conformation into which a grid can be inserted alongthe long diagonal. Transfer container 12 can be secured, for example bymeans of a screw 14, in a suitable orientation in which openings 13advantageously extend with their long diagonal parallel to the aforesaidvertical plane.

After deposition of the sectioned material, the forceps is rotated aboutits longitudinal axis by rotating receptacle 56 (FIG. 2) 180°, with theresult that grid 6 assumes a vertical position and points verticallydownward. By displacement via the three axes of the manipulator, thegrid can now be brought into one of the openings 13 of container 12 anddeposited there by an opening operation using slide 11. Transfercontainer 12 having the grid is closed off with a cover (FIG. 2) andtaken out of the chamber.

After deposition of the grid into the transfer container, forceps 10 isremoved from receptacle 56 (FIG. 2) and can be loaded, outside thedevice, with a new grid for a further sectioning operation.

While one or more embodiments of the present invention have beendescribed above, it should be understood that any and all equivalentrealizations of the present invention are included within the scope andspirit thereof. Thus, the embodiments presented herein are provided byway of example only.

1. A microtome system comprising: a cryomicrotome having a working spaceand a sectioning device comprising a preparation holder and a knife edgethat are guidable past one another inside the working space during asectioning operation in which the preparation holder retains apreparation to thereby produce thin sections of the preparation; amicromanipulator; and a tool for retaining a specimen support, whereinthe tool is positionable by the micromanipulator to position thespecimen support proximate the knife edge during the sectioningoperation, and wherein a portion of the micromanipulator that controlsposition of the tool is located outside the working space.
 2. Themicrotome system according to claim 1, wherein the micromanipulator isconfigured to retain the specimen support substantially stationaryduring the sectioning operation.
 3. The microtome system according toclaim 1, comprising a cooling chamber that surrounds the working spaceso that an opening from an area outside the cooling chamber to theworking space permits access of the tool to the working space, andwherein the portion of the micromanipulator that controls position ofthe tool is located outside the cooling chamber.
 4. The microtome systemaccording to claim 3, wherein the micromanipulator is mounted on thecooling chamber.
 5. The microtome system according to claim 1, whereinthe micromanipulator is fixedly joined to the cryomicrotome.
 6. Themicrotome system according to claim 1, wherein the micromanipulatorcomprises a three-axis positioning device.
 7. The microtome systemaccording to claim 1, wherein the micromanipulator comprises areceptacle, and the tool comprises a forceps received by the receptacle.8. The microtome system according to claim 7, wherein the forceps iselongated and has a tip that is bent with respect to a longitudinaldimension of the forceps.
 9. The microtome system according to claim 8,wherein the forceps tip is bent at an angle of approximately 45° withrespect to the longitudinal dimension.
 10. The microtome according toclaim 1, wherein the tool is elongated and is retained in themicromanipulator so that the tool is rotatable about a longitudinal axisof the tool.
 11. The microtome system as in claim 8, wherein the tool isretained in the micromanipulator so that the forceps is rotatable aboutan axis along the longitudinal dimension.
 12. The microtome systemaccording to claim 1, comprising a transfer container that comprises atleast one receiving opening for a specimen support is replaceablyinsertable in the working space, and wherein the portion of themicromanipulator that controls position of the tool controls the toolthrough a range of motion sufficient to move the specimen supportretained by the tool to the receiving opening of a transfer containerinserted in the working space.
 13. The microtome system according toclaim 1, wherein one or more axes of motion of the micromanipulator aremotorized.